Paracingulin is an M(r) 150-160 kDa cytoplasmic protein of vertebrate epithelial tight and adherens junctions and comprises globular head, coiled-coil rod, and globular tail domains. Unlike its homologous tight junction protein cingulin, paracingulin has been implicated in the control of junction assembly and has been localized at extrajunctional sites in association with actin filaments. Here we analyze the role of paracingulin domains, and specific regions within the head and rod domains, in the function and localization of paracingulin by inducible overexpression of exogenous proteins in epithelial Madin Darby canine kidney (MDCK) cells and by expression of mutated and chimeric constructs in Rat1 fibroblasts and MDCK cells. The overexpression of the rod + tail domains of paracingulin perturbs the development of the tight junction barrier and Rac1 activation during junction assembly by the calcium switch, indicating that regulation of junction assembly by paracingulin is mediated by these domains. Conversely, only constructs containing the head domain target to junctions in MDCK cells and Rat1 fibroblasts. Furthermore, expression of chimeric cingulin and paracingulin constructs in Rat1 fibroblasts and MDCK cells identifies specific sequences within the head and rod domains of paracingulin as critical for targeting to actin filaments and regulation of junction assembly, respectively. In summary, we characterize the functionally important domains of paracingulin that distinguish it from cingulin.